Application of selenium-enriched probiotics in preparation of preparation for treating fluoxetine-induced liver injury

Abstract

The invention provides an application of selenium-rich probiotics in preparation of a preparation for treating fluoxetine-induced liver injury, and relates to the technical field of biological medicine, the application is characterized in that Se-BL is obtained by reducing sodium selenite through ascorbic acid and modifying the surface of bifidobacterium longum, and nano-scale selenium dots are formed. The preparation plays a role through dual mechanisms: active oxygen (ROS) is directly removed, an Nrf2 pathway is activated, and the liver oxidation resistance (GSH, SOD) is improved; the traditional Chinese medicine composition is capable of inhibiting NF-kappa B inflammation signal channels, reducing proinflammatory factors (TNF-alpha and IL-6) and synchronously regulating intestinal flora so as to enhance the intestinal barrier function. Experiments prove that the traditional Chinese medicine composition can remarkably reduce the level of serum transaminase (ALT / AST / ALP) and relieve liver tissue necrosis and lipid peroxidation damage (MDA), the dosage form of the traditional Chinese medicine composition is an oral preparation, and each dose of the traditional Chinese medicine composition contains 1 * 10 <-1 > * 10 <-1 > CFU of Se-BL viable bacteria. The selenium-modified probiotics are used for antagonizing hepatotoxicity of mental drugs for the first time, and an innovative therapy is provided for fluoxetine-induced drug-induced liver injury (DILI) and acute hepatic failure (ALF).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and more particularly relates to application of selenium-rich probiotics in preparation of a preparation for treating fluoxetine-induced liver injury. BACKGROUND

[0002] Fluoxetine is the most widely used selective serotonin reuptake inhibitor (SSRI) in clinical application at present, which can selectively inhibit serotonin transporter, block the reuptake of 5-hydroxytryptamine at the presynaptic membrane, prolong and increase the effect of 5-hydroxytryptamine, and thus produce an antidepressant effect. It is widely used in clinic due to its high tolerance and few side effects. It is a first-line prescription drug for the treatment of depression and other neuropsychiatric diseases. However, fluoxetine has a long half-life, is mainly metabolized in the liver into active product norfluoxetine, and is excreted through urine. Its metabolism process involves inhibition of cytochrome P450 enzymes, which may cause drug interactions. Although the clinical safety is high, long-term use can cause adverse reactions such as anxiety, insomnia, gastrointestinal reactions, metabolic disorders and sexual dysfunction. More seriously, due to the particularity of the disease and the long medication period (antidepressant treatment usually requires more than 6 months of drug intake), long-term use has serious hepatotoxicity, leading to liver injury and even acute liver failure (ALF). At present, there is a lack of specific treatment for fluoxetine-induced liver injury or acute liver failure, and patients often progress rapidly due to the disease, leading to multiple organ failure, and even need to rely on liver transplantation to maintain life, but due to the scarcity of donor resources and high medical costs, it brings serious challenges to the public health system.

[0003] The intestinal tract is not only an organ of digestion and absorption, but also an important immune system. The microflora, immune cells and their metabolites in the intestinal tract enter the blood circulation through the intestinal barrier, affecting the immune response, inflammatory response and metabolic process of the liver. Studies have shown that the intestinal microflora or its metabolites can affect the progression of various liver diseases, for example, in acetaminophen-administered mice, the abundance of beneficial bacteria Lactobacillus in the intestinal tract is significantly reduced, while Bifidobacterium longum reverses these phenomena; in addition, after antibiotic intervention of the intestinal tract, the incidence of liver cancer is significantly reduced. Therefore, the gut-liver axis is an important biological pathway, and the use of probiotics to manipulate the intestinal microflora may provide a new safe treatment for liver diseases.

[0004] Fluoxetine-induced liver injury and ALF are generally believed to be related to the excessive free radicals generated during its metabolism, which induce oxidative stress and cause liver tissue damage by activating inflammatory mediators. However, recent studies have shown that intestinal flora also plays an important role in fluoxetine-induced hepatotoxicity. For example, long-term fluoxetine treatment affects the microbial subpopulation and has a negative impact on health. Specifically, the abundance of Porphyromonadaceae and Alistipes in fecal microorganisms increases, and these genera are associated with some neuroinflammation; in addition, intestinal flora disorders can reduce the efficacy of fluoxetine, and the drug resistance of E. coli isolated from patients using antidepressants is significantly higher; in addition, compared with drug treatment alone, probiotic combination therapy increases the bacterial abundance of Lactobacillus to stabilize the balance of intestinal microbiota, maintain intestinal barrier function, reduce immune and inflammatory responses, and further reduce liver injury.

[0005] The probiotic BL (Bifidobacterium longum) used in this project is a kind of long bifidobacterium. Studies have shown that BL protects mice from APAP-induced liver injury by regulating the Nrf2 pathway; it has also been reported that BL improves acute liver injury in mice induced by lipopolysaccharide (LPS) through the nuclear factor-κB pathway, in addition, BL also regulates intestinal flora, enhances intestinal barrier function, and reduces the ectopic and LPS release of E. coli.

[0006] Although there have been many studies on the use of probiotics to prevent or treat drug-induced liver injury, their antioxidant capacity is limited, they rely on indirect antioxidant effects of microbial metabolites (such as butyric acid), and they lack enzyme systems (such as GPx) that directly scavenge ROS, so their protective effect is insufficient in drug-induced liver injury. As a core cofactor of glutathione peroxidase and thioredoxin reductase (TrxR), selenium (Se) directly scavenges ROS and activates the Nrf2 pathway to enhance antioxidant defense; it also inhibits NF-κB inflammatory signals and regulates T cell differentiation to reduce liver immune damage; it maintains energy metabolism homeostasis by protecting mitochondrial membrane integrity and electron transport chain function. Compared to ordinary probiotics, which are more inclined to daily regulation of mild intestinal microecological imbalance, selenium-enriched probiotics (Se-BL) have better preventive and therapeutic effects.

[0007] Therefore, it is necessary to study the potential of selenium-enriched probiotics (Se-BL) in improving fluoxetine-induced hepatotoxicity through a rat model of fluoxetine and to explore its possible mechanisms from a multi-omics perspective, in order to provide a new direction for the prevention and treatment of fluoxetine-induced liver injury and ALF. SUMMARY

[0008] To solve the above technical problems, the application provides the use of selenium-enriched probiotics in the preparation of a preparation for treating fluoxetine-induced liver injury, to solve the above problems.

[0009] Use of selenium-enriched probiotics in the preparation of a preparation for treating fluoxetine-induced liver injury, wherein the Se-BL is formed by reducing sodium selenite with ascorbic acid to form selenium nanoparticles and is modified on the surface of Bifidobacterium longum, and the preparation method of the Se-BL comprises: (a) resuspending Bifidobacterium longum in a 0.25-1 mM sodium selenite solution, and incubating at room temperature for 1-3 hours; (b) adding ascorbic acid dropwise to a final concentration of 1-5 mM, and reacting for 1-3 hours; (c) centrifuging and washing to obtain Se-BL loaded with selenium nanoparticles on the surface, wherein the drug reduces the levels of serum ALT, AST and ALP induced by fluoxetine by 40%-60%, reduces the levels of serum TNF-α and IL-6 by 30%-60%, restores the GSH content in the liver to 60%-90% of the normal level, increases the SOD activity by 1.5-2.2 times, and reduces the MDA level by 40%-70%, and the drug is an oral preparation containing 1×10 8 -1×10¹ 0 CFU, administered once a day for 7 days.

[0010] A pharmaceutical composition for preventing or treating fluoxetine-induced liver injury, comprising Se-BL and a pharmaceutical carrier, wherein the carrier is selected from at least one of a freeze-drying protective agent, a prebiotic or an enteric coating material.

[0011] Preferably, the Se-BL enhances antioxidant defense by activating the Nrf2 pathway and reduces inflammatory response by inhibiting the NF-κB pathway, the Se-BL cooperatively improves the gut-liver axis function by regulating the abundance of intestinal flora, enhancing intestinal barrier integrity and inhibiting LPS release, and the drug is used for treating fluoxetine-induced acute liver failure (ALF), and the characterization of the Se-BL is confirmed by transmission electron microscopy (TEM) and element mapping to confirm the distribution density of selenium nanoparticles and the morphology of the bacteria.

[0012] A biomarker detection method for fluoxetine-induced liver injury, wherein the therapeutic effect of Se-BL is evaluated by monitoring the levels of serum ALT, AST, TNF-α and liver GSH and MDA.

[0013] Compared with the prior art, the present application has the following beneficial effects:

[0014] Synergistic effect: selenium nanoparticles directly scavenge ROS, and probiotics regulate the flora, and the double mechanism reduces the liver injury markers (ALT / AST decreases by 40%-60%).

[0015] Tissue repair: significantly reduces the necrosis area of liver cells (confirmed by H&E staining) and improves the disorder of liver lobule structure.

[0016] Clinical application advantage: oral preparation avoids liver first-pass effect, live bacteria colonize the intestinal tract to achieve long-term protection, and is suitable for patients who take fluoxetine for a long time. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the synthetic route of Se-BL and TEM Mapping characterization figure of the present application;

[0018] Figure 2 is the histological section result of rat liver of the present application;

[0019] Figure 3 is the blood biochemical result of Se-BL on fluoxetine-induced liver injury of the present application;

[0020] Figure 4 is the improvement result of Se-BL on serum inflammatory markers caused by fluoxetine of the present application;

[0021] Figure 5 is the improvement result of Se-BL on liver oxidative stress caused by fluoxetine of the present application. DETAILED DESCRIPTION

[0022] The embodiments of the present application will be further described below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present application, but cannot be used to limit the scope of the present application.

[0023] Please refer to Figures 1-5 , the present application provides the application of selenium-rich probiotics in the preparation of preparations for treating fluoxetine-induced liver injury. Se-BL forms nanoselenium points by reducing sodium selenite with ascorbic acid and is modified on the surface of Bifidobacterium longum. The preparation method of Se-BL comprises: (a) resuspending Bifidobacterium longum in a 0.25-1 mM sodium selenite solution, incubating at room temperature for 1-3 hours; (b) adding ascorbic acid dropwise to a final concentration of 1-5 mM, and reacting for 1-3 hours; (c) centrifuging and washing to obtain Se-BL loaded with selenium points on the surface. The drug reduces the serum ALT, AST and ALP levels induced by fluoxetine by 40%-60%, reduces the serum TNF-α and IL-6 levels by 30%-60%, restores the liver GSH content to 60%-90% of the normal level, increases the SOD activity by 1.5-2.2 times, and reduces the MDA level by 40%-70%. The drug is an oral preparation, each dose contains 1×10 8 -1×10 0 CFU, administered once a day for 7 days.

[0024] A pharmaceutical composition for preventing or treating fluoxetine-induced liver injury, comprising Se-BL and a pharmaceutical carrier, wherein the carrier is selected from at least one of a lyophilization protective agent, a prebiotic or an enteric coating material.

[0025] Se-BL enhances antioxidant defense by activating Nrf2 pathway and inhibits inflammatory response by suppressing NF-κB pathway, Se-BL improves gut-liver axis function by regulating gut microbiota abundance, enhancing intestinal barrier integrity and inhibiting LPS release, the drug is used for treating fluoxetine-induced acute liver failure (ALF), the characterization of Se-BL confirms the distribution density of selenium points and the morphology of bacteria by transmission electron microscopy (TEM) and element Mapping.

[0026] A biomarker detection method for fluoxetine-induced liver injury, the therapeutic effect of Se-BL is evaluated by monitoring serum ALT, AST, TNF-α and liver GSH, MDA levels.

[0027] Figure 1 Preparation and characterization of Se-BL

[0028] (A) Preparation process of Se-BL, reaction was carried out at room temperature in Na2SeO3 and ascorbic acid respectively; (B) TEM characterization of Se-BL; (C) Mapping characterization of Se-BL. White arrows indicate large selenium points, white arrows indicate small selenium points.

[0029] Figure 2 Histological sections of rats

[0030] (A) Control rats; (B) Fluoxetine treatment group (10 mg / kg); (C) BL (1×109 CFU) + fluoxetine treatment group (10 mg / kg); (D) Se-BL (1×109 CFU) + fluoxetine treatment group (10 mg / kg). Red arrows indicate areas of hepatocyte necrosis, color significantly lighter; black arrows indicate disordered liver lobule structure.

[0031] Figure 3 Effect of Se-BL on serum enzyme markers of liver function induced by fluoxetine

[0032] (A) ALT: alanine aminotransferase; (B) AST: aspartate aminotransferase; (C) ALP: serum alkaline phosphatase. Data are expressed as SEM ± mean (N=6). **P<0.01, ***P<0.001, ****P<0.0001.

[0033] Figure 4 Effect of Se-BL on serum inflammatory markers of fluoxetine-treated rats

[0034] (A) TNF-α: tumor necrosis factor; (B) IL-6: interleukin-6. These values are expressed in pg / mL. Data are expressed as SEM ± mean (N=6). *P<0.05, ***P<0.001, ****P<0.0001.

[0035] Figure 5 Effect of Se-BL on the antioxidant status of liver tissue of rats treated with fluoxetine

[0036] (A) GSH: reduced glutathione; (B) SOD: superoxide dismutase; (C) MDA: malondialdehyde. Data are expressed as SEM ± mean (N = 6). **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0037] I. Synthesis and characterization of Se-BL

[0038] Logarithmic phase B. longum (BL) (OD600nm=0.6) was centrifuged at 4000 rpm for 5 min and washed with 5 mL of PBS three times to remove residual medium. The bacterial pellet was resuspended in 0.3 mM sodium selenite solution (Na2SeO3) and incubated for 120 min with stirring. Subsequently, the Vc solution was added to the mixture and the reaction was stirred at room temperature for 2 h. PBS solution was washed three times to remove unreacted sodium selenite and Vc. The size and morphology of Se-BL were characterized by scanning electron microscopy (TEM).

[0039] II. Verification of the activity of Se-BL

[0040] A stable model of fluoxetine-induced liver injury was constructed based on existing reports. To determine the effect of Se-BL on fluoxetine-induced liver injury, rats were gavaged with probiotic preparation (1 x 109) or an equivalent amount of PBS and 10 mg / kg of fluoxetine for a total of 7 days. At the end of the experiment, the rats were euthanized and samples were taken.

[0041] Hepatocyte necrosis and inflammatory infiltration were evaluated by hematoxylin and eosin (H&E) staining.

[0042] The degree of liver injury was evaluated by serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP).

[0043] The severity of liver inflammation was evaluated by serum TNF-α and IL-6.

[0044] The degree of oxidative stress in the liver was evaluated by GSH, SOD, and MDA levels in liver homogenate.

[0045] Effect verification:

[0046] I. Synthesis and characterization of Se-BL

[0047] The size of probiotics is usually micron level, which cannot be observed by common optical microscope. TEM is a powerful tool combining high-resolution imaging and elemental analysis, which is suitable for multi-scale characterization of nanomaterials, biological samples and so on. The distribution of Se element on the surface of probiotics can be clearly observed by combining with elemental / structural mapping, just as Figure 1 The TEM mapping results show that the Se dots (green part) reduced by ascorbic acid are densely distributed, and will be gathered into several large Se dots. The shape of the probiotics can be roughly observed, which proves that most of the Se dots are adsorbed on the surface of the probiotics.

[0048] II. Activity verification of Se-BL

[0049] H&E staining of liver The main mode of liver injury was proved by morphology and staining. The liver injury of each group was shown in Figure 2 , and it can be seen from Figure 2 that the necrosis area (light part) of the liver in the Se-BL+fluoxetine group was significantly lower than that in the fluoxetine group, while the BL++fluoxetine group had no obvious improvement. Histologically, it is proved that Se-BL treatment can significantly improve the liver injury induced by fluoxetine.

[0050] The levels of liver injury markers ALT, AST and ALP in each group were shown in Figure 3 It has been reported in the literature that the type of drug-induced liver injury caused by fluoxetine is mainly hepatocyte injury, in which ALT mainly exists in the cytoplasm of hepatocytes, and is released into the blood in large quantities after hepatocyte membrane damage, which specifically reflects the injury of liver parenchyma; while AST exists in liver (mitochondria and cytoplasm), myocardium and skeletal muscle, and its elevation may reflect hepatocyte necrosis or systemic injury. Compared with AST and ALT indicators, ALP mainly reflects cholestasis or bile duct injury, and its change is significantly lower than that of the former, and Figure 3 data just verify this point. It can be seen that the liver injury markers ALT and AST in the fluoxetine group and the Se-BL+fluoxetine group are significantly increased, but the indicators in the Se-BL+fluoxetine group are lower than those in the fluoxetine group. This result proves from the biochemical level that Se-BL gavage can partially alleviate drug-induced liver injury. Compared with the BL+fluoxetine group, the liver enzyme level in the Se-BL+fluoxetine group is closer to that in the control group, indicating that Se-BL has better anti-liver injury function.

[0051] Inflammatory response is an important reason for fluoxetine-induced liver injury, and the detection of TNF-α and IL-6 has important pathological significance. TNF-α aggravates liver necrosis by activating hepatocyte apoptosis and promoting neutrophil infiltration, while IL-6 amplifies the inflammatory response and inhibits liver regeneration, and its elevated serum level can be used as an early warning indicator. Figure 4Results showed that fluoxetine administration caused a significant decrease in GSH levels in rat liver and induced a significant decrease in antioxidant enzyme SOD in rat liver. The determination of malondialdehyde (MDA) can be used as a marker of lipid peroxidation in the liver. Compared with the control group of rats, fluoxetine-induced rats had a significantly increased level of lipid peroxidation. At the same time, Se-BL significantly reduced the level of lipid peroxidation in the liver of rats induced by fluoxetine.

[0052] During the metabolism of fluoxetine, reactive oxygen species (ROS) are produced to consume glutathione (GSH). The activity of superoxide dismutase (SOD) will increase in the early stage of compensation, but will decrease due to ROS overload and functional inhibition after continuous damage, leading to imbalance of antioxidant defense system; and probiotics can reverse this phenomenon. Figure 5 Results showed that fluoxetine administration caused a significant decrease in GSH levels in rat liver and induced a significant decrease in antioxidant enzyme SOD in rat liver. The determination of malondialdehyde (MDA) can be used as a marker of lipid peroxidation in the liver. Compared with the control group of rats, fluoxetine-induced rats had a significantly increased level of lipid peroxidation. At the same time, Se-BL significantly reduced the level of lipid peroxidation in the liver of rats induced by fluoxetine.

[0053] Example 1: Preparation and characterization of Se-BL

[0054] OD 600 =0.6 of long bifidobacterium bacteria solution, centrifugal washing and resuspension in 0.3mM sodium selenite solution, stirring incubation for 2 hours. Add ascorbic acid dropwise to a final concentration of 3mM, centrifuge to remove free ions after 2 hours of reaction. TEM and element Mapping show that selenium points (diameter 5-20nm) are densely distributed on the surface of the bacteria.

[0055] Example 2: Liver injury treatment experiment

[0056] Fluoxetine liver injury rat model was constructed (10mg / kg gavage, 7 days). The experimental group was given Se-BL (1×10 9 Results:

[0057] Histology: The necrosis area of hepatocytes in the Se-BL group was significantly reduced compared with the fluoxetine group;

[0058] Biochemical indicators: ALT and AST levels decreased by more than 50%, TNF-α and IL-6 decreased by 45%-55%;

[0059] Oxidative stress: GSH returned to normal level of 85%, MDA decreased by 65%.

[0060] Example 3: Pharmaceutical preparation

[0061] Se-BL was mixed with trehalose (lyophilization protective agent) and fructooligosaccharides (prebiotics) to make enteric-coated capsules. Each capsule contains 5×10 9CFU, once daily orally.

[0062] Embodiments of the application are presented for the purpose of illustration and description, and not by way of limitation or limitation of the application. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments were chosen and described in order to best explain the principles of the application and its practical application, and to enable others skilled in the art to understand the application in order to design various embodiments with various modifications as suitable for the particular use contemplated.

Claims

1. Use of selenium-enriched probiotic bacteria for the preparation of a medicament for the treatment of fluoxetine-induced liver injury, characterized in that: The Se-BL is formed by reducing sodium selenite with ascorbic acid to form selenium nanoparticles and is modified on the surface of B. longum.

2. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury, characterized in that, The preparation method of the Se-BL comprises: (a) resuspending B. longum in a 0.25-1 mM sodium selenite solution and incubating at room temperature for 1-3 hours; (b) adding ascorbic acid dropwise to a final concentration of 1-5 mM and reacting for 1-3 hours; (c) centrifuging and washing to obtain Se-BL loaded with selenium nanoparticles on the surface.

3. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury, characterized in that, The drug reduces the serum ALT, AST and ALP levels induced by fluoxetine by 40%-60%, reduces the serum TNF-α and IL-6 levels by 30%-60%, restores the liver GSH content to 60%-90% of the normal level, increases the SOD activity by 1.5-2.2 times, and reduces the MDA level by 40%-70%.

4. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury, characterized in that, The medicament is an oral preparation containing 1 x 10 8 CFU of Se-BL viable bacteria per dose, administered once daily for 7 days. 0 CFU, administered once daily for 7 days.

5. A pharmaceutical composition for preventing or treating fluoxetine-induced liver injury, characterized by, The Se-BL comprises the Se-BL according to any one of claims 1-4 and a pharmaceutical carrier selected from at least one of a freeze-drying protective agent, a prebiotic or an enteric coating material.

6. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury. The Se-BL enhances antioxidant defense by activating the Nrf2 pathway and reduces inflammatory response by inhibiting the NF-κB pathway.

7. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury. The Se-BL improves the function of the intestinal-liver axis by regulating the abundance of intestinal flora, enhancing intestinal barrier integrity and inhibiting the release of LPS.

8. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury. The drug is used for treating acute liver failure (ALF) induced by fluoxetine.

9. Use of the selenium-enriched probiotic bacteria according to claim 1 for the preparation of a medicament for the treatment of fluoxetine-induced liver injury. The characterization of the Se-BL confirms the distribution density of selenium nanoparticles and the morphology of the bacteria by transmission electron microscopy (TEM) and element Mapping.

10. A method for detecting a biomarker of liver injury of fluoxetine, characterized by, The therapeutic effect of the Se-BL is evaluated by monitoring the serum ALT, AST, TNF-α and liver GSH and MDA levels.

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